1

Interferon-β induces loss of spherogenicity and overcomes therapy resistance

of glioblastoma stem cells

Caroline Happold1, Patrick Roth1, Manuela Silginer1, Ana-Maria Florea4, Katrin

Lamszus3, Karl Frei2, Rene Deenen4, Guido Reifenberger4,5, Michael Weller1

1Laboratory of Molecular Neuro-Oncology, Department of Neurology, and

2Department of Neurosurgery, University Hospital Zurich, Frauenklinikstrasse 26, and

Neuroscience Center Zurich, 8091 Zurich, Switzerland; 3Laboratory for Brain Tumor

Biology, Department of Neurosurgery, University Hospital Hamburg-Eppendorf,

Martinistrasse 52, 20246 Hamburg, Germany; 4Department of Neuropathology (A.F.,

G.R.) and Center for Biological and Medical Research (R.D.), Heinrich Heine

University, Düsseldorf, Germany, 5German Cancer Consortium (DKTK), German

Cancer Research Center (DKFZ), Heidelberg, Germany

*Correspondence: Prof. Michael Weller, Department of Neurology, University

Hospital Zurich, Frauenklinikstrasse 26, 8091 Zurich, Switzerland, Tel.: +41 (0)44

255 5500, Fax: +41 (0)44 255 4507, E-mail: Michael.Weller@usz.ch

Running title: Anti-glioma activity of interferon-β

Keywords: glioma, interferon, MGMT, stem cell, temozolomide, irradiation

This work was supported by a grant from the Swiss National Science Foundation

(31003A_130122) to M.Weller.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

2

Abbreviations

CNPase, 2', 3'-cyclic nucleotide 3'-phosphodiesterase; DMSO, dimethylsulfoxide;

EGF, epidermal growth factor; FGF, fibroblast growth factor; FCS, fetal calf serum;

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acidic

protein; GIC, glioma-initiating cells; GO, gene ontology; IFN, interferon; IFNAR,

interferon receptor; IL, interleukin; JAK, Janus kinase; LTL, long-term cell line; MFL,

Mega-FAS-ligand; MGMT, O6-methylguanine DNA methyltransferase; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered

saline; PI, propidium iodide; RMA, robust multi-array analysis; RT-PCR, reverse

transcriptase PCR; SFI, specific fluorescence intensity; sh, small hairpin; si, small

interfering; STAT, Signal Transducers and Activators of Transcription; STRING,

Search Tool for the Retrieval of Interacting Genes/Proteins; STS, staurosporine;

TMZ, temozolomide; TP53, tumor protein p53; TNFSF10/TRAIL, tumor necrosis

factor-related apoptosis-inducing ligand; XAF-1, XIAP-associated factor 1; XIAP, X-

linked inhibitor of apoptosis

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

3

ABSTRACT

Glioblastoma is the most common malignant brain tumor in adults and characterized

by a poor prognosis. Glioma cells expressing O6-methylguanine DNA

methyltransferase (MGMT) exhibit a higher level of resistance towards alkylating

agents, including the standard of care chemotherapeutic agent, temozolomide (TMZ).

Here, we demonstrate that long-term glioma cell lines (LTL) as well as glioma-

initiating cell lines (GIC) express receptors for the immune modulatory cytokine

interferon (IFN)-β and respond to IFN-β with induction of STAT-3 phosphorylation.

Exposure to IFN-β induces a minor loss of viability, but strongly interferes with sphere

formation in GIC cultures. Further, IFN-β sensitizes LTL and GIC to TMZ and

irradiation. RNA interference confirmed that both IFN-β receptors, R1 and R2, are

required for IFN-β-mediated sensitization, but that sensitization is independent of

MGMT or TP53. Most GIC lines are highly TMZ-resistant mediated by MGMT

expression, but nevertheless susceptible to IFN-β sensitization. Gene expression

profiling following IFN-β treatment revealed strong up-regulation of IFN-β-associated

genes including a proapoptotic gene cluster, but did not alter stemness-associated

expression signatures. Caspase activity and inhibition studies revealed the

proapoptotic genes to mediate glioma cell sensitization to exogenous death ligands

by IFN-β, but not to TMZ or irradiation, indicating distinct pathways of death

sensitization mediated by IFN-β. Thus, IFN-β is a potential adjunct to glioblastoma

treatment that may target the GIC population. IFN-β operates independently of

MGMT-mediated resistance, classical apoptosis-regulatory networks and stemness-

associated gene clusters.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

4

INTRODUCTION

Glioblastomas are characterized by infiltrative growth and resistance to cell death

induction. Despite multimodal therapy, tumor progression occurs inevitably, and

survival remains in the range of months (1-3). Early therapy failure is associated with

the expression of O6-methylguanine-DNA-methyltransferase (MGMT), a DNA repair

protein that accounts for glioma cell resistance by counteracting the effects of

alkylating chemotherapy (4). MGMT has become an important molecular marker and

is now implemented in clinical diagnostics as predictive biomarker for benefit from

alkylating chemotherapy and clinical outcome (5-7). Patients with a non-methylated

MGMT promoter are prone to therapy failure with standard alkylating chemotherapy,

and to date, effective approaches for this large group of patients, comprising more

than half of all glioblastoma patients, are still lacking, including dose intensification of

TMZ (8).

Recently, a subfraction of glioma cells exhibiting stem cell-like properties (stem-like

glioma cells), referred to as glioma-initiating cells (GIC), has been identified (9, 10).

GIC are thought to have the ability of self-renewal, tumor initiation and pluripotency,

and have been proposed to account for the ultimately lethal nature of glioblastoma.

They may contribute to therapy resistance in vivo and therefore promote tumor

progression. The value of GIC cultures and their MGMT status as a model to study

resistance to TMZ in glioblastoma has remained controversial. Among a panel of 20

GIC lines, some were sensitive to TMZ, which was associated with low MGMT

protein levels, but not MGMT promoter methylation, and the MGMT promoter status

was thus not strongly predictive of response to TMZ (11). High TMZ sensitivity of GIC

cultures lacking MGMT expression has been described in vitro (12). Finally, the

frequency of MGMT promoter-methylated alleles in glioblastomas may range from

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

5

10% to 90%, but methylated alleles were enriched in GIC cultures (13). Considering

the limited activity of current standards of care, new approaches should take into

account this novel target cell population.

IFN-β, a member of the interferon class I family, exerts numerous functions for

cellular differentiation, cell growth and immune responses. IFN-β signaling is

mediated through binding to a type II cytokine receptor, involving heterodimerization

of 2 IFN-α/β receptor subunits, IFNAR-1 and IFNAR-2. Down-stream signalling

pathways involve the Janus kinase/signal Transducers and Activators of

Transcription (JAK/STAT) pathway (14) and lead to accumulation of MxA protein, a

GTPase interfering with the cytoskeletal structure (15, 16). MxA induction is an

established marker for responsiveness to IFN-α/β (17, 18).

IFN-β has gained great clinical impact in the treatment of multiple sclerosis. Its safety

profile in patients with brain disease has therefore been firmly established. Moreover,

IFN-α/β were the first agents to show a significant survival benefit in randomized

trials in malignant melanoma (19). The mechanisms mediating antitumor effects of

IFN may involve direct cytostatic, anti-angiogenic or immune modulatory activities.

IFN-β has been reported to sensitize human glioma cell lines to TMZ in a TP53- and

MGMT-dependent manner (20, 21). These reports, however, were based on findings

in T98G and U251MG, both being TP53 mutant cell lines (22, 23). Moreover, IFN-β

has been attributed anti-stem cell properties in glioma models (24). Finally, clinical

trials exploring the addition of IFN-β to radiotherapy plus TMZ have been initiated and

interpreted as promising (25, 26). These data prompted us to evaluate the potential

role of IFN-β as a component of future, innovative treatment approaches for

glioblastoma in more detail. In brief, we demonstrate profound anti-GIC activity of

IFN-β as well as IFN-β-mediated sensitization to TMZ and irradiation, but exclude

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

6

MGMT and TP53 as primary mediators of these effects. IFN-β exposure induces

characteristic IFN-β-associated gene clusters, but does not alter the stemness

signature of GIC. A proapoptotic gene signature induced by IFN-β is functionally

relevant, but mediates neither anti-GIC properties nor sensitivity to TMZ or

radiotherapy.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

7

MATERIALS AND METHODS

Materials and cell lines

The human U87MG and T98G glioma cell lines, K562 leukemia cells and MCF-7

breast adenocarcinoma cells were purchased from the American Type Culture

Collection (Rockville, MD, USA). The other long-term cell lines (LTL) were kindly

provided by N. de Tribolet (Lausanne, Switzerland). LNT-229 cells cultured in our

laboratory express TP53 wild-type transcriptional activity (23). The generation of

LNT-229 cells depleted of TP or overexpressing MGMT has been described (23, 27).

MGMT-silenced LN-18 cells were generated by transfection with pSUPER puro

encoding MGMT short hairpin (sh) RNA using Metafectene Pro transfection reagent

(Biontex Laboratories GmbH, Martinsried, Germany) (28). The GIC cell lines GS-2,

GS-5, GS-7, GS-8 and GS-9 have been characterized elsewhere (29). LTL were

cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf

serum (FCS), 2 mM glutamine and penicillin (100 IU/ml) / streptomycin (100 μg/ml).

GS sphere cultures were maintained in Neurobasal Medium® supplemented with 2%

B-27 supplement, 1% GlutaMAX, 20 ng/ml epidermal growth factor, 20 ng/ml

fibroblast growth factor and 32 IE/ml heparin. K-562 cells were maintained in RPMI-

1640 supplemented with 1 mmol/L sodium pyruvate (Gibco Life Technologies) and

10% fetal calf serum. TMZ was provided by Schering-Plough (Kenilworth, NJ, USA).

A stock solution of TMZ at 200 mM was prepared in dimethylsulfoxide (DMSO).

Recombinant IFN-β1b was purchased from AbD Serotec (Dusseldorf, Germany),

reconstituted to a concentration of 1,000,000 IU/ml with distilled water. The following

antibodies were used: STAT-3 and phospho-STAT-3, Caspase-3 and LC3A/B from

Cell Signaling (Boston, MA, USA); glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) from Everest Biotech (Oxfordshire, UK); tumor necrosis factor-related

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

8

apoptosis inducing ligand (TRAIL) and XIAP-associated factor 1 (XAF-1) from Santa

Cruz (Santa Cruz, CA, USA); caspase 8 from Enzo Life Science (Framingdale, NY,

USA); Glial fibrillary acidic protein (GFAP) from Dako (Carpinteria, CA, USA); Nestin

from Zytomed Systems GmbH (Berlin, Germany); β-III tubulin from Abcam

(Cambridge, UK). Mega-Fas-ligand (MFL) was provided by Topotarget (Copenhagen,

Denmark). The MxA antibody was kindly provided by O. Haller, MD and G. Kochs,

PhD (Department of Virology, University of Freiburg, Germany). All other reagents,

including anti- 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) antibody, O6-

benzylguanine (O6-BG) and salinomycin, were purchased from Sigma (St. Louis, MO,

USA). For irradiation experiments, cells were irradiated in a Co-radiation source

(Gebrüder Sulzer, Thermische Energiesysteme, 60-Co, Winterthur, Switzerland) at 1,

3 and 5 Gy at 24 h after IFN-β exposure.

Polymerase chain reaction

Total RNA was prepared using the NucleoSpin System (Macherey-Nagel AG,

Önsingen SO, Switzerland) and transcribed using random primers (Bioconcept/NEB,

Bioconcept, Allschwil, Switzerland) and Superscript II reverse transcriptase

(Invitrogen, Carlsbad, CA, USA). For reverse transcriptase PCR, the conditions were:

35 cycles, 94°C/45 sec, specific annealing temperature (54°C for IFNAR-1, 52°C for

IFNAR-2, 51°C for GAPDH) /45 sec, 72°C/1 min. For real-time (quantitative) RT-

PCR, cDNA amplification was monitored using SYBRGreen chemistry on the 7300

Real time PCR System (Applied Biosystems, Zug, Switzerland). The conditions were:

40 cycles, 95°C/15 sec, 60°C/1 min. Data analysis was done using the ΔΔCT method

for relative quantification. The following specific primers were used: GAPDH fwd: 5'-

CTCTCTGCTCCTCCTGTTCGAC-3', GAPDH rv: 5'-TGAGCGATGTGGCTCGGCT-

3'; IFNAR-1 fwd: 5'-TAT GCT GCG AAA GTC TTC TTG AG-3’; IFNAR-1 rv: 5'-TCT

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

9

TGG CTA GTT TGG GAA CTG TA-3’; IFNAR-2 fwd: 5'-TCT TGA GGC AAG GTC

TCG CTA-3’; IFNAR-2 rv: 5'-CAG GGA TGC ACG CTT GTA ATC-3’; XAF1 fwd: 5’-

AGC AGG TTG GGT GTA CGA TG-3’; XAF1 rv: 5’-TGA GCT GCA TGT CCA GTT

TG-3’; TRAIL fwd: 5’-TGC GTG CTG ATC GTG ATC TTC-3’ ; TRAIL rv: 5’-GCT CGT

TGG TAA AGT ACA CGT A-3’.

Flow cytometry, cell cycle and viability assays

Differentiated glioma cells and sphere cultures were detached respectively

dissociated using Accutase (PAA Laboratories, Pasching, Austria) and blocked with

2% FCS in phosphate-buffered saline (PBS). The cells were incubated for 30 min on

ice using the following PE-labeled antibodies: anti–IFNAR-1 or anti-IFNAR-2

antibodies (PBL interferon source, Piscataway, NJ, USA) for IFNAR experiments and

anti-CD133/2-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) for stem cell marker

experiments. Flow cytometry was performed with a CyAn® flow cytometer (Beckman

Coulter, Nyon, Switzerland). Signal intensity was calculated as the ratio of the mean

fluorescence of the specific antibody and the isotype control antibody (specific

fluorescence index). Dead cells were gated out. For some analyses, cells were

permeabilized by Fix/Perm Buffer Set (Lucerna Chem AG, Biolegend, Luzern,

Switzerland). For analysis of cell death, cells were grown in six-well plates, incubated

with IFN-β at 150 IU/ml for 24 h, washed in PBS and allowed to grow for 48 h.

Annexin (Anx) V-fluorescein isothiocyanate (1:100) and propidium iodide (PI) (50

µg/ml) were added, and fluorescence in a total of 10,000 events (cells) per condition

was recorded in a CyAn flow cytometer. AnxV- or PI-positive cells were counted as

dead cells, the remaining cells were designated the surviving cell fraction. In some

experiments, loss of viability was also confirmed by trypan blue dye exclusion.

Autophagic cell death was assessed by immunoblot and immunostaining. In brief, for

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

10

the latter, the cells were exposed to IFN-β as indicated and Cytospin samples were

prepared, fixed in 4% PFA, blocked in TBS containing 0.2% Triton X100, 5% goat

serum and 5% horse serum, and exposed to the primary LC3A/B antibody (Cell

Signaling, Boston, MA, USA) overnight at 4°C and 1 h at room temperature for

secondary antibodies.

Clonogenicity assays

For LTL, clonogenicity assays were performed by seeding 100 cells (LNT-229, LN-

18) per well in 96-well plates, allowed to adhere overnight, and exposed to IFN-β at

150 IU/ml for 24 h in fresh medium. After removal of IFN-β, the cells were exposed to

TMZ at the indicated concentrations for 24 h in serum-free medium, followed by an

agent-free observation period for 7-14 days in serum-containing medium. Cell density

was assessed by crystal violet staining. For sphere cultures, the cells were seeded at

500 cells per well in neurobasal medium and treated consecutively as indicated

above in neurobasal medium. Lower cell numbers resulted in inefficient sphere

formation. Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay. Initially, we confirmed that crystal violet

assay and MTT assay were good surrogate markers of the number of colonies

respectively spheres in these assays, but easier to standardize for large scale

concentration response analyses.

Immunoblot analysis

The cells were treated as indicated and lysed in lysis buffer containing 50 mM Tris-

HCl, 120 mM NaCl, 5 mM EDTA and 0.5% NP-40. Protein (20 µg/lane) was

separated on 10% acrylamide gels. After transfer to nitrocellulose (Biorad, Munich,

Germany), the blots were blocked in Tris-buffered saline containing 5% skim milk and

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

11

0.05% Tween 20 and incubated overnight at 4°C with primary antibodies and 1 h at

room temperature for secondary antibodies. Visualization of protein bands was

accomplished using horseradish peroxidase-coupled secondary antibodies (Santa

Cruz Healthcare, Santa Cruz, CA, USA) and enhanced chemiluminescence (Pierce /

Thermo Fisher, Madison, WI, USA).

Immunocytochemistry and immunofluorescence microscopy

For stemness experiments, the cells were exposed to IFN-β (150 IU/ml) for 48 h and

cytospin samples were prepared, fixed in 4% PFA and blocked in either blocking

solution (Candor Bioscience GmbH, Wangen, Germany) for GFAP, β-III tubulin or

CNPase, or PBS containing 10% swine serum and 0.3% Triton X for nestin, and

exposed to the primary antibody overnight at 4°C and 30 min at room temperature for

secondary antibodies. Detection was performed with DAB detection systems (Dako)

for 5 min at room temperature, and slides were mounted in Eukitt (Sigma-Aldrich).

Counterstaining was performed with hemalum. For IFNAR receptor staining, cytospin

samples of LTL or GIC were prepared as described above. Staining was performed

as suggested by the manufacturer, using IFNAR-1 antibody from antibodies-

online.com (Atlanta, GA, USA) and IFNAR-2 antibody from Abcam (Cambridge, UK).

RNA interference-mediated gene silencing

For transient transfections, 2.5 × 105 glioma cells were seeded in a six-well plate and

transfected with 100 nM of specific or scrambled control small interfering (si) RNA,

using Metafectene Transfection reagent (Biontex). siRNA was purchased from

Dharmacon / Thermo Fisher (Chicago, IL, USA) using siGENOME SMARTpool

targeting human IFNAR-1 and human IFNAR-2. Samples were collected 48 h post

transfection and processed for further treatment.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

12

Reporter assay

Dual luciferase/renilla assays were carried out with co-transfection of 150 ng of the

specific reporter construct and 20 ng of the renilla reniformis-CMV (pRL-CMV) control

plasmid (Promega, Madison, WI, USA). Luciferase activity was normalized to

constitutive renilla activity. The pGL2-Luc MGMT construct (30) was a kind gift from

Dr. S. Mitra (Sealy Center for Molecular Science and Department of Human

Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston,

TX, USA). The TP53-Luc construct has been described elsewhere (31).

Caspase activity assay

Cells were seeded at a density of 1000 cells/well in a six-well-plate and treated as

described above with either IFN-β and TMZ alone or in combination with or without

ZVAD-fmk, with either MFL (Mega-FAS-ligand) or staurosporine as positive control.

Cells were incubated in lysis buffer (25 mM Tris/HCL, 60 mM NaCl, 2.5 mM EDTA,

0.25% NP40) for 10 min, and the substrate Ac-DEVD-amc was added at a

concentration of 20 μM. Fluorescence was assessed at 360 nm wavelength every 15

min until extinction of fluorescence (32).

Microarray-based gene expression profiling

Technical details are provided in the supplementary methods. Array records are

deposited under GEO accession number GSE53213.

Data analysis

Data are representative of experiments performed three times with similar results.

Where indicated, analysis of significance was performed using the two-tailed

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

13

Student’s t-test. Synergy of irradiation or TMZ and IFN-β was assessed by the

fractional product method (33) where indicated and differences of 10% of observed

versus predicted (additive) effect were considered synergistic. For the analysis of

functional gene interactions, the Search Tool for the Retrieval of Interacting

Genes/Proteins (STRING) Version 9.05 at http://string-db.org (34) was used. Highest

confidence settings were applied, integrating combined scores higher than 0.900.

Cluster analysis was performed by application of the Markov Cluster algorithm

(MCL). Disconnected nodes were hidden from the image.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

14

RESULTS

IFNAR are expressed in human glioma cells

In total, 12 LTL and 5 GIC were assessed for mRNA expression of IFNAR-1 and -2

by RT-PCR. All lines expressed transcripts for both receptor subunits (Fig. 1A).

IFNAR-1, in contrast to IFNAR-2, was not detected at the surface on any glioma cell

line by flow cytometry (Fig. 1B, left panel), but was detected by immunostaining (Fig.

1B, right panel). Permeabilization of the cells or scraping of cells instead of using

accutase did not produce a specific signal for IFNAR-1 on flow cytometry either.

Specific fluorescence intensity (SFI) values for IFNAR-2 above 3 were never seen in

GIC, but in all LTL (data not shown).

IFN-β signalling in glioma cells involves MxA and STAT-3

To ensure that IFN-β induces classical signalling pathways in glioma cells, we

investigated whether STAT-3, the proposed signal transducer in response to IFN-β in

gliomas, and the IFN-α/β-regulated protein MxA, a classical marker for cellular

responsiveness to IFN-β, were induced. Immunoblot confirmed that STAT-3

phosphorylation levels increased shortly after exposure to IFN-β, with a peak at 30-

60 min, with no relevant concentration dependency between 50 and 500 IU/ml, in

LTL and GIC (Fig. 1C). Likewise, the exposure to IFN-β led to a concentration-

dependent increase of MxA protein in LNT-229 (Fig. 1D, upper row) or LN-18 cells

(data not shown), in a time-dependent manner (Fig. 1D, lower row), with first marked

elevation of protein levels at a concentration of 150 IU/ml at 24 h.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

15

IFN-β induces cell cycle arrest in glioma cells and causes sphere disruption in

GIC

Within the concentration and time ranges of our experiments, IFN-β induced a

reduction of G1 cells in all cell lines, associated with increases in S cells or G2/M cells

or both. Moreover, there was an increase in sub-G1 cells indicating minor cell death

induction (Fig. 2A). A moderate induction of necrotic rather than apoptotic cell death

was confirmed by AnxV/PI flow cytometry, defined by an increase of the PI-positive

fraction (Fig. 2B). Cellular staining and immunoblot for autophagy were negative in

response to IFN-β (Fig. 2C, D).

We also assessed a possible modulation of spherogenicity of GIC by IFN-β. When

exposed to 150 IU/ml IFN-β, singularized GIC cultures showed a reduced sphere

formation at 1,000 – 10,000 cells per well; lower cell numbers did not result in

efficient sphere formation, independent of IFN-β exposure (Fig. 2E). Conversely,

exposure of fully formed spheres to 150 IU/ml IFN-β led to sphere disaggregation,

with lesser and significantly smaller spheres remaining after 2 weeks of culture, as

assessed by sphere count and absorbance (Fig. 2F). The single cells detaching from

the sphere were viable during the first days after sphere disaggregation, but

underwent cell death after a week, as assessed by trypan blue assay (data not

shown).

To assess the possible impact of IFN-β on stem cell differentiation, we analysed GS-

2, 5, 7 and 9 cells for changes in the expression of different stem cell markers. GS-2

and GS-9 cells expressed CD133, but expression remained unchanged after

exposure to 150 IU/ml IFN-β for 48 h. GS-5 and GS-7 did not express CD133 (Fig.

2G). Moreover, IFN-β-treated GS-2, GS-5 and GS-9 cells showed no changes of

GFAP, nestin, β-III-Tubulin or CNPase staining (shown for GS-2) (Fig. 2H).

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

16

Requirement for IFNAR-1 and IFNAR-2 for IFN-β-mediated sensitization to

irradiation and TMZ

To verify the role of IFNAR-1 and IFNAR-2 for IFN-β-mediated signal transduction

and sensitization to TMZ and irradiation, we silenced the expression of both

receptors by siRNA. Efficacy of gene silencing was assessed by RT-PCR for IFNAR-

1, which could not be assessed by flow cytometry (Fig. 1), and by RT-PCR and flow

cytometry for IFNAR-2 (Fig. 3A). In the setting of TMZ exposure (Fig. 3B) or

irradiation (Fig. 3C), silencing of IFNAR-1 or -2 or both attenuated the sensitizing

effect of IFN-β in LNT-229 cells and LN-18 cells, confirming a role for IFNAR-

mediated signal transduction pathways in the IFN-β-induced sensitization process.

IFNAR-2 gene silencing alone did not abrogate the effect of IFN-β on TMZ activity to

the same extent as IFNAR-1 gene silencing alone.

Response to TMZ, irradiation and IFN in GIC lines

Since GIC are proposed to be the major source of tumor relapse and resistance, we

next focused on these models. All investigated GIC, except GS-9, expressed MGMT

and were highly resistant to TMZ (Supplementary Note 1, Fig. S1). IFN-β had no

major effect on cell density of LTL cells, but a significant concentration-dependent

impact of IFN-β alone was observed in GIC cells (Fig. 4A), as expected from the data

shown in Fig. 2F. To assess whether IFN-β also partially overcomes TMZ resistance

of GIC, we pre-exposed the cells to increasing concentrations of IFN-β (Fig. 4B, left

panel), or to 150 IU/ml IFN-β for increasing periods of time (Fig. 4B, right panel), prior

to treatment with TMZ at EC50 concentrations. All investigated GIC lines showed

sensitization to TMZ after pre-exposure to IFN-β. Sensitization was significant in all

cell lines when treated at a concentration of 150 IU/ml and for an exposure time of 24

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

17

h. Exposure of GS-2 cells to IFN-β for 24 h before administration of TMZ induced a

strong growth-inhibiting effect on the sphere cultures (Fig. 4C). IFN-β-treated cells

showed a decrease in clonogenic survival exceeding 50% compared to TMZ only-

treated cells, and exhibited a progressive loss of the sphere structure (see also Fig.

2D). Similar effects, albeit with less impressive disaggregation, were observed for

GS-5, GS-7 and GS-9 spheres, where cooperative effects were most prominent

when IFN-β was given with TMZ at approximately EC50 concentrations.

In the setting of irradiation experiments, GIC cells were not more resistant than LTL,

with GS-2 displaying the strongest radioresistance of the panel, and GS-5 the

weakest, possibly because of the TP53 wild-type status of this cell line (29) (Fig. 4D).

Pre-exposure to increasing concentrations of IFN-β followed by low dose irradiation

at 1 Gy resulted at least in additive inhibition of clonogenic survival. Again, in the

rather radioresistant GS-2, a significant impact of IFN-β alone was observed (Fig. 4E,

left panel); preexposure to IFN-β before irradiation led to a reduction of cell density

fulfilling criteria of synergy, with an additional reduction of cell density of almost 50%

when exposed to 150 IU/ml of IFN-β. GS-5 and GS-7 displayed synergistic effects at

150 IU/ml IFN-β; no synergy was determined in GS-9 (Fig. 4E).

Gene expression analyses

Microarray-based gene expression profiling demonstrated IFN-β-dependent

significant differential regulation of 509 genes after 6 h of IFN-β treatment in all three

glioma cells lines investigated. Gene expression profiling after 24 h revealed

significant differential regulation of 522 genes. The combined analysis, comparing the

6 h and 24 h data, resulted in an overlap of 132 differentially expressed genes upon

exposure to IFN-β in all three cell lines. In addition to the statistical analysis, where

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

18

IFN-β treatment effects were studied globally across all three cell lines, the impact of

IFN-β was assessed individually for every single cell line. Single analysis probe lists

of all three cell lines were compared at 6 h and 24 h, respectively (Fig. S2), defining

overlapping lists of regulated genes as well as lists of genes exclusively regulated in

each individual cell line. Multiple transcripts known to be regulated by IFN-β were

found, including STAT1, interferon regulatory factors (IRF) and Mx1 (MxA) (Table

S1). Moreover, we noted that a number of genes predicted to promote apoptotic

signaling were induced by IFN-β, (Table S1), with an overlap of 6 genes annotated to

both gene groups. Submission of the differentially expressed IFN-regulated genes to

the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) identified a

single main cluster of genes with strong interaction profile (Fig. S3A). Submission of

the differentially expressed apoptosis-related genes revealed 3 main clusters, one

cluster involving tumor necrosis factor-related apoptosis-inducing ligand (TRAIL /

TNFSF10)-interacting genes (yellow), one involving XIAP-associated factor 1 (XAF1)-

interacting genes (blue), both connected via a third cluster centered around STAT1

(green) (Fig. S3B). Induction of TRAIL and XAF1 was confirmed by RT-PCR and

immunoblot analysis (Fig. 5). As we had observed differential reaction of the LTL cell

line LNT-229 and the GIC cell lines GS-2 and GS-9 in clonogenic growth assays after

exposure to IFN-β alone, we performed a gene ontology (GO) analysis of the genes

exclusively regulated (FC > 2) in LNT-229, or the GIC lines (Fig. S2, peripheral

regions), and identified several down-regulated genes involved in proliferation and

cell growth, as well as up-regulated genes involved in negative regulation of

proliferation or cell cycle arrest (Table S2). Notably, more genes were regulated in

the GIC lines, with 2 main clusters of negative regulators of proliferation interacting

mainly through JUN (blue cluster) and IL-8, IL-6 and JAK2 (red cluster) (Fig. S3C).

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

19

Altered expression of apoptosis regulating genes does not mediate enhanced

clonogenic cell death induced by TMZ or irradiation

To define a role for altered expression of apoptotic machinery genes in the biological

effects of IFN-β, we examined whether IFN-β alone or when combined with different

death stimuli promoted caspase activation under the conditions that induced

synergistic inhibition of clonogenic survival (Figure 4). As a positive control, we used

MFL since we previously observed IFN-α-induced sensitization to CD95L although

the mechanism remained unclear then (35). MFL, but neither IFN-β nor TMZ,

induced the cleavage of caspases 8 or 3 or DEVD-amc-cleaving caspase activity

(Fig. 6A, B, C). Similar results were obtained for irradiation assays (data not shown).

Preexposure to IFN-β did not result in caspase processing in cells exposed to TMZ

either. Cell death in the investigated settings was confirmed by trypan blue dye

exclusion (Fig. 6D). Finally, we performed clonogenic cell death assays similar to

those in Figure 4 in the absence or presence of the broad spectrum caspase

inhibitor, zVAD-fmk, to explore whether caspase inhibition attenuated or abrogated

the effects in clonogenic cell death assays of IFN-β or TMZ alone or in combination

(Fig. 6E). These assays confirmed the absence of a role for caspases, either upon

single agent exposure or combination. The biological activity of zVAD-fmk under

these conditions was confirmed by the inhibition of death receptor-mediated

apoptosis as previously reported (36).

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

20

DISCUSSION

Even after the implementation of TMZ as the first active chemotherapeutic agent

when combined with standard radiotherapy, glioblastomas remain a major challenge

in the field of neuro-oncology as they often relapse early and follow an invariably fatal

clinical course. The latter is also true for the subgroup of patients with MGMT

promoter methylation who derive most benefit from TMZ (4, 6). Large efforts have

been made to improve prognosis by investigating new agents, either at recurrence,

but recently more often early in development already in the paradigm of concomitant

and adjuvant TMZ plus radiotherapy (3, 37, 38).

Here we asked whether the immune modulatory cytokine, IFN-β, acts on glioma LTL

and GIC lines, and sensitizes for the anti-clonogenic effects of TMZ or irradiation. We

detected the expression of IFNAR-1 and -2 mRNA in all glioma cells, making them a

potential target for IFN-β (Fig. 1A). Although detection of IFNAR-1 protein turned out

to be challenging and was possible by immunofluorescence microscopy only, but not

by flow cytometry, the biological effects of IFNAR-1 gene silencing were prominent:

siRNA-mediated knockdown of the single IFNAR-1 chain inhibited IFN-β-mediated

sensitization (Fig. 3), thereby confirming its biological function. Silencing of the

IFNAR-2 chain alone inhibited IFN-β signalling only to a moderate level whereas

silencing of IFNAR-1 was additionally required to abort the intracellular signal

cascade enough to lead to a highly significant reduction in inhibition of clonogenic

survival after IFN-β exposure (Fig. 3B,C). This may be due to the fact that, whilst

IFNAR-2 holds only ligand-binding capacities, IFNAR-1, mainly inducing the

intracellular signalling cascades, has also been described to have weak ligand

binding activity, too, and may interact with other proteins in the absence of IFNAR-2

(39, 40). Alternatively, although both siRNA pools reduced receptor mRNA

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

21

expression to a similar extend (Fig. 3A), differential protein stability may account for

different biological efficiency of gene silencing in these experiments. Responsiveness

of the cells was assessed by monitoring the expression of target proteins of the IFN-β

signaling pathways (Fig. 1D).

STAT-3 has been shown to be involved in antiproliferative functions when induced by

IFN-α/β in human Daudi cells (41) and, in glioma cells, may act as an IFN-β-induced

tumor inhibitor through negative modulation of miR-21 (42). Conversely, there is also

accumulating evidence that STAT-3 is a driver of the malignant phenotype of

glioblastoma (43). In our study, STAT-3 was uniformly phosphorylated in both LTL

and GIC cell lines shortly after exposure to IFN-β (Fig. 1C). Thus, the biological

consequences of STAT-3 phosphorylation may be context-dependent and need to be

interpreted in the natural course or the therapeutic setting in which they are

observed.

Since GIC cells cultured under sphere conditions are considered to be a more

resistant subpopulation of glioma cells (44-46), we characterized cell cycle

progression, cell death induction and sphere formation capacity of GIC in the

presence of IFN-β. Similar to LTL, we observed a cell cycle arrest with a reduced G1

phase and an increase of the sub-G1 population, reflecting a moderate increase in

the necrotic fraction on AnxV/PI flow cytometry (Fig. 2A,B). Exposure of singularized

cells isolated from sphere cultures to IFN-β led to reduced sphere formation;

moreover, fully formed spheres exposed to IFN-β for 24 h disaggregated in the first

days (Fig. 2E,F), and the cells underwent delayed cell death after more than a week

as assessed by trypan blue dye exclusion (data not shown). This sphere disruption

effect with delayed cell death induction appears to be a major effect of IFN-β on GIC

survival and to play a more important role than early classical apoptotic pathways

that were not shown to be activated in the first 48 h post exposure in AnxV/PI flow

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

22

cytometry. Accordingly, Affymetrix array data demonstrated up-regulation of several

apoptosis-related genes (Table S1, Fig. S3B), but ZVAD-fmk-controlled clonogenicity

assays or DEVD-amc-cleaving-assays did again not confirm an induction of classical

caspase-determined apoptotic pathways (Fig. 6C,E).

We demonstrated that IFN-β facilitated the loss of clonogenicity induced by the

standard chemotherapeutic agent for glioblastoma, TMZ. Since a sensitizing

mechanism mediated through MGMT and TP53 had been described (20), we

assessed IFN-β effects on a panel of cells with a heterogeneous pattern of MGMT

and TP53 status (Supplementary Note 2, Fig.S4A). In our experimental settings,

neither TP53 nor the MGMT status was important for the sensitizing effects of IFN-β.

Specifically, we did not observe an induction of TP53 expression in cells with TP53

wild-type transcriptional activity (LNT-229) or a down-regulation of MGMT expression

in MGMT-expressing cells (LN-18) (Supplementary Note 2, Fig. S4C,D), nor did we

observe MGMT- or TP53-associated changes in clonogenic survival assays, where

sensitization by IFN-β was achieved independent of MGMT or TP53 status

(Supplementary Note 2, Fig. S4A,B). Finally, Affymetrix chip analyses did neither

detect induction of TP53 nor down-regulation of MGMT in the cell line expressing the

respective gene products (TP53: LNT-229, GS-2; MGMT: GS-2; data not shown).

The sensitization in the presence of MGMT is particularly attractive in view of the

urgent clinical need for novel strategies for the majority of glioblastoma patients with

tumors without MGMT promoter methylation. Moreover, we confirmed that

preexposure to IFN-β sensitized different LTL to the anti-clonogenic effects of single

doses of irradiation, too (Fig. S4B), and that these effects were mediated by the

known receptors of IFN-β, IFNAR-1 and IFNAR-2 (Fig. 3).

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

23

GIC lines, which were confirmed here to be highly resistant to TMZ due to MGMT

expression, appeared to be a special target to IFN-β which impaired sphere formation

when administered alone, in the absence of major cell death induction (Fig. 2).

Accordingly, we observed a growth-inhibiting effect of IFN-β in GIC cultures even

when applied as a single agent (Fig. 4A), even in MGMT-expressing and highly TMZ-

resistant models (Fig. 4B-E). These chemosensitizing effects of IFN-β were

concentration- and time-dependent, with significant results assessed at a

concentration of 150 IU/ml, and a 24 h application (Fig. 4B, E). Of note, the molecular

pattern in GIC cells was also diverse with regard to TP53 status (29), confirming that

TP53 is not a major mediator of IFN-β-induced sensitization in glioma cells.

All glioma models investigated here were sensitized at concentrations of IFN-β

reached in the serum in clinical settings following intravenous application, where

ranges of over 1000 IU/ml were reported at doses of 18x106 IE i.v. in healthy

volunteers (47-49). IFN-β has been applied intravenously before in several clinical

studies and was shown to be well tolerated without significant side effects (50),

making it an interesting candidate for adjuvant glioblastoma therapy, close to

practice. Thus, the present laboratory evidence, together with the wealth of data on

the safety and tolerability of IFN-β in patients with multiple sclerosis, justifies further

clinical trials of IFN-β in combination with radiotherapy and TMZ in patients with

newly diagnosed glioblastoma, specifically those patients suffering from MGMT-

unmethylated tumors.

Acknowledgement

The authors would like to thank Dr. Sankar Mitra and Dr. Kishor Bhakat for providing

the MGMT reporter plasmid, as well as Jasmin Buchs, Nadine Lauinger and Silvia

Dolski for excellent technical assistance.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

24

Conflict of interest statement: C.H. has received honoraria for advisory board

activity from MSD. P.R. has received honoraria for advisory board activity from MSD,

Roche and Molecular Partners. M.W. received research support from Bayer,

Antisense Pharma, Merck Serono and Roche and honoraria for advisory board and

lecture activities from Merck Serono, MSD, Roche and Magforce. G.R. received

honoraria for advisory board activities from Merck Serono and Roche. M.S., A. M. F.,

K.L., R.D. and K.F. declare no conflict of interest.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

25

References

1. Johnson DR, O'Neill BP. Glioblastoma survival in the United States before and

during the temozolomide era. J Neurooncol 2012;107:359-64.

2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et

al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N

Engl J Med 2005;352:987-96.

3. Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med 2008;359:492-

507.

4. Hegi ME, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, et al.

MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med

2005;352:997-1003.

5. Weller M, Pfister SM, Wick W, Hegi ME, Reifenberger G, Stupp R. Molecular

neuro-oncology in clinical practice: a new horizon. Lancet Oncol 2013;14:e370-9.

6. Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W, et

al. MGMT promoter methylation in malignant gliomas: ready for personalized

medicine? Nat Rev Neurol 2010;6:39-51.

7. Wick W, Platten M, Meisner C, Felsberg J, Tabatabai G, Simon M, et al.

Temozolomide chemotherapy alone versus radiotherapy alone for malignant

astrocytoma in the elderly: the NOA-08 randomised, phase 3 trial. Lancet Oncol

2012;13:707-15.

8. Gilbert MR, Wang M, Aldape KD, Stupp R, Hegi ME, Jaeckle KA, et al. Dose-

dense temozolomide for newly diagnosed glioblastoma: a randomized phase III

clinical trial. J Clin Oncol 2013;31:4085-91.

9. Park DM, Rich JN. Biology of glioma cancer stem cells. Mol Cells 2009;28:7-

12.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

26

10. Venere M, Fine HA, Dirks PB, Rich JN. Cancer stem cells in gliomas:

identifying and understanding the apex cell in cancer's hierarchy. Glia 2011;59:1148-

54.

11. Blough MD, Westgate MR, Beauchamp D, Kelly JJ, Stechishin O, Ramirez AL,

et al. Sensitivity to temozolomide in brain tumor initiating cells. Neuro Oncol

2010;12:756-60.

12. Beier D, Rohrl S, Pillai DR, Schwarz S, Kunz-Schughart LA, Leukel P, et al.

Temozolomide preferentially depletes cancer stem cells in glioblastoma. Cancer Res

2008;68:5706-15.

13. Sciuscio D, Diserens AC, van Dommelen K, Martinet D, Jones G, Janzer RC,

et al. Extent and patterns of MGMT promoter methylation in glioblastoma- and

respective glioblastoma-derived spheres. Clin Cancer Res 2011;17:255-66.

14. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells

respond to interferons. Annu Rev Biochem 1998;67:227-64.

15. Goetschy JF, Zeller H, Content J, Horisberger MA. Regulation of the

interferon-inducible IFI-78K gene, the human equivalent of the murine Mx gene, by

interferons, double-stranded RNA, certain cytokines, and viruses. J Virol

1989;63:2616-22.

16. Haller O, Kochs G. Interferon-induced mx proteins: dynamin-like GTPases with

antiviral activity. Traffic 2002;3:710-7.

17. Holzinger D, Jorns C, Stertz S, Boisson-Dupuis S, Thimme R, Weidmann M, et

al. Induction of MxA gene expression by influenza A virus requires type I or type III

interferon signaling. J Virol 2007;81:7776-85.

18. von Wussow P, Jakschies D, Hochkeppel HK, Fibich C, Penner L, Deicher H.

The human intracellular Mx-homologous protein is specifically induced by type I

interferons. Eur J Immunol 1990;20:2015-9.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

27

19. Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ, Borden EC, Blum RH.

Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the

Eastern Cooperative Oncology Group Trial EST 1684. J Clin Oncol 1996;14:7-17.

20. Natsume A, Ishii D, Wakabayashi T, Tsuno T, Hatano H, Mizuno M, et al. IFN-

beta down-regulates the expression of DNA repair gene MGMT and sensitizes

resistant glioma cells to temozolomide. Cancer Res 2005;65:7573-9.

21. Natsume A, Wakabayashi T, Ishii D, Maruta H, Fujii M, Shimato S, et al. A

combination of IFN-beta and temozolomide in human glioma xenograft models:

implication of p53-mediated MGMT downregulation. Cancer Chemother Pharmacol

2008;61:653-9.

22. Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, Diserens AC, et al. Frequent

co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in

human glioma cell lines. Brain Pathol 1999;9:469-79.

23. Wischhusen J, Naumann U, Ohgaki H, Rastinejad F, Weller M. CP-31398, a

novel p53-stabilizing agent, induces p53-dependent and p53-independent glioma cell

death. Oncogene 2003;22:8233-45.

24. Williams RF, Sims TL, Tracey L, Myers AL, Ng CY, Poppleton H, et al.

Maturation of tumor vasculature by interferon-beta disrupts the vascular niche of

glioma stem cells. Anticancer Res 2010;30:3301-8.

25. Motomura K, Natsume A, Kishida Y, Higashi H, Kondo Y, Nakasu Y, et al.

Benefits of interferon-beta and temozolomide combination therapy for newly

diagnosed primary glioblastoma with the unmethylated MGMT promoter: A

multicenter study. Cancer 2011;117:1721-30.

26. Watanabe T, Katayama Y, Yoshino A, Fukaya C, Yamamoto T. Human

interferon beta, nimustine hydrochloride, and radiation therapy in the treatment of

newly diagnosed malignant astrocytomas. J Neurooncol 2005;72:57-62.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

28

27. Hermisson M, Klumpp A, Wick W, Wischhusen J, Nagel G, Roos W, et al. O6-

methylguanine DNA methyltransferase and p53 status predict temozolomide

sensitivity in human malignant glioma cells. J Neurochem 2006;96:766-76.

28. Maurer GD, Tritschler I, Adams B, Tabatabai G, Wick W, Stupp R, et al.

Cilengitide modulates attachment and viability of human glioma cells, but not

sensitivity to irradiation or temozolomide in vitro. Neuro Oncol 2009;11:747-56.

29. Gunther HS, Schmidt NO, Phillips HS, Kemming D, Kharbanda S, Soriano R,

et al. Glioblastoma-derived stem cell-enriched cultures form distinct subgroups

according to molecular and phenotypic criteria. Oncogene 2008;27:2897-909.

30. Biswas T, Ramana CV, Srinivasan G, Boldogh I, Hazra TK, Chen Z, et al.

Activation of human O6-methylguanine-DNA methyltransferase gene by

glucocorticoid hormone. Oncogene 1999;18:525-32.

31. Wischhusen J, Melino G, Weller M. p53 and its family members -- reporter

genes may not see the difference. Cell Death Differ 2004;11:1150-2.

32. Wagenknecht B, Schulz JB, Gulbins E, Weller M. Crm-A, bcl-2 and NDGA

inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8

processing. Cell Death Differ 1998;5:894-900.

33. Webb JL. Enzyme and metabolic inhibitors. 1st ed New York: Academic press

1963.

34. Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, et al.

STRING v9.1: protein-protein interaction networks, with increased coverage and

integration. Nucleic acids research 2013;41:D808-15.

35. Roth W, Wagenknecht B, Dichgans J, Weller M. Interferon-alpha enhances

CD95L-induced apoptosis of human malignant glioma cells. J Neuroimmunol

1998;87:121-9.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

29

36. Glaser T, Wagenknecht B, Groscurth P, Krammer PH, Weller M. Death

ligand/receptor-independent caspase activation mediates drug-induced cytotoxic cell

death in human malignant glioma cells. Oncogene 1999;18:5044-53.

37. Weller M, Stupp R, Hegi M, Wick W. Individualized targeted therapy for

glioblastoma: fact or fiction? Cancer J 2012;18:40-4.

38. Wick W, Weller M, Weiler M, Batchelor T, Yung AW, Platten M. Pathway

inhibition: emerging molecular targets for treating glioblastoma. Neuro Oncol

2011;13:566-79.

39. Baychelier F, Nardeux PC, Cajean-Feroldi C, Ermonval M, Guymarho J, Tovey

MG, et al. Involvement of the Gab2 scaffolding adapter in type I interferon signalling.

Cell Signal 2007;19:2080-7.

40. Cutrone EC, Langer JA. Contributions of cloned type I interferon receptor

subunits to differential ligand binding. FEBS Lett 1997;404:197-202.

41. Yang CH, Murti A, Pfeffer LM. STAT3 complements defects in an interferon-

resistant cell line: evidence for an essential role for STAT3 in interferon signaling and

biological activities. Proc Natl Acad Sci U S A 1998;95:5568-72.

42. Ohno M, Natsume A, Kondo Y, Iwamizu H, Motomura K, Toda H, et al. The

modulation of microRNAs by type I IFN through the activation of signal transducers

and activators of transcription 3 in human glioma. Mol Cancer Res 2009;7:2022-30.

43. Abou-Ghazal M, Yang DS, Qiao W, Reina-Ortiz C, Wei J, Kong LY, et al. The

incidence, correlation with tumor-infiltrating inflammation, and prognosis of

phosphorylated STAT3 expression in human gliomas. Clin Cancer Res

2008;14:8228-35.

44. Bao S, Wu Q, Sathornsumetee S, Hao Y, Li Z, Hjelmeland AB, et al. Stem cell-

like glioma cells promote tumor angiogenesis through vascular endothelial growth

factor. Cancer Res 2006;66:7843-8.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

30

45. Li Z, Wang H, Eyler CE, Hjelmeland AB, Rich JN. Turning cancer stem cells

inside out: an exploration of glioma stem cell signaling pathways. J Biol Chem

2009;284:16705-9.

46. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al.

Identification of human brain tumour initiating cells. Nature 2004;432:396-401.

47. Buchwalder PA, Buclin T, Trinchard I, Munafo A, Biollaz J. Pharmacokinetics

and pharmacodynamics of IFN-beta 1a in healthy volunteers. J Interferon Cytokine

Res 2000;20:857-66.

48. Chiang J, Gloff CA, Yoshizawa CN, Williams GJ. Pharmacokinetics of

recombinant human interferon-beta ser in healthy volunteers and its effect on serum

neopterin. Pharm Res 1993;10:567-72.

49. Salmon P, Le Cotonnec JY, Galazka A, Abdul-Ahad A, Darragh A.

Pharmacokinetics and pharmacodynamics of recombinant human interferon-beta in

healthy male volunteers. J Interferon Cytokine Res 1996;16:759-64.

50. Larocca AP, Leung SC, Marcus SG, Colby CB, Borden EC. Evaluation of

neutralizing antibodies in patients treated with recombinant interferon-beta ser. J

Interferon Res 1989;9 Suppl 1:S51-60.

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

31

Figure legends

Fig. 1. IFNAR-1 and -2 expression and responsiveness to IFN-β in human LTL

and GIC. A,B. The cells were assessed for expression of IFNAR-1 and IFNAR-2

mRNA by PCR with GAPDH as control (A) and of protein by cell surface flow

cytometry (B, left panel; representative profiles for K562 as a positive control and

LNT-229, LN-18, LN-308, GS-2 and GS-9 GIC cells; black curve: isotype control;

grey curve: IFNAR antibody), or immunofluorescence staining (B, right panel; MCF-7

and K562 were used as control). C. Phosphorylation levels of STAT-3 in a

concentration- (left) and time-dependent (right) manner, shown representatively for

LNT-229 cells (upper row), and for GS-7 and GS-9 GIC in a time-dependent manner

(lower row). D. Responsiveness to IFN-β was assessed through detection of MxA

protein in a concentration- (upper row) and time-dependent (lower row) manner,

shown representatively for LNT-229 cells.

Fig. 2. Assessment of IFN-β effects on GIC cells. A,B. LTL or GIC cells were

exposed to IFN-β at 150 IU/ml for 24 h, cultured for an additional 48 h in serum-

enriched medium, and flow cytometric cell cycle analysis (A) or AnxV/PI flow

cytometry (B) were performed. Cell distributions are shown as bar graphs (striped:

sub-G1, black: G1, white: S, grey: G2/M). Cell lines in B match those shown in A

above. C. LNT-229 or GS-2 cells were exposed to IFN-β (150 IU/ml), ddH2O aqua

(negative control) or salinomycin (SAL) (4 µM) (positive control) for 24 h. D. LNT-229

and GS-2 cells were treated as in C for protein lysates and assessed for LC3 A/B

cleavage. GAPDH was used as control. E. The effect of IFN-β (150 IU/ml, 24 h) on

sphere formation over a range of 5 – 10,000 single cells was assessed by sphere

count and is shown for GS-2 (black square, IFN-β; white diamond, vehicle). Data are

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

32

expressed as mean ± SEM (n=6) (***p

33

effect of IFN-β is represented as delta of IFN-β-exposed versus vehicle-exposed

cells.

Fig. 4. GIC sensitization to TMZ and irradiation by IFN-β

A. LTL or GIC cells were exposed to IFN-β in a concentration-dependent manner and

clonogenic survival was assessed by crystal violet (LTL) or MTT (GIC) assay. B. LTL

or GIC cells were exposed to IFN-β in a concentration-dependent (left) or time-

dependent (right) manner before a 24 h pulse of TMZ in an EC50 concentration

range. Clonogenic survival was assessed by crystal violet or MTT assay. C. GS-2,

GS-5, GS-7 or GS-9 cells pre-exposed to IFN-β (150 IU/ml, 24 h) were treated with

TMZ as shown in Fig. 3A and allowed to grow in neurobasal medium for 2 weeks

before the assessment of clonogenicity by MTT assay (control: white bar; IFN-β:

black bar). Data are expressed as mean ± SEM (*p

34

TRAIL were assessed by RT-PCR for RNA and by immunoblot for protein at 6 h and

24 h after exposure to IFN-β in increasing doses (time points as assessed by micro-

array profiling), in LNT-229 (left) and GS-2 (right).

Fig. 6. Role of apoptosis-regulatory genes in IFN-β-induced sensitization.

A, B. Immunoblots were performed after exposure of LNT-229 (left panel) or GS-2

(right panel) to IFN-β for 6 h (upper row) or 24 h (lower row) and the levels of

caspase 8 and 3 were assessed either for IFN-β exposure alone, TMZ alone, or in

combination as described. MFL and STS were used as a positive control (MFL: 1

μg/ml; STS: 1μM). C. DEVD-amc cleaving assays were performed after 6 h of

exposure (shown for 2h fluorescence measurement). D. Cell death was assessed by

trypan blue exclusion after 6 h of exposure to IFN-β at 150 or 300 IU/ml, IFN-β +

TMZ (10 μM for LNT-229, 500 μM for GS-2), or MFL and STS as positive controls, as

described in C; ddH2O was used as negative control (white bars, alive cells; black

bars, dead cells; normalized to 100% cells). E. Clonogenic cell death assays were

performed for LNT-229 and GS-2 and analyzed by crystal violet staining (for LNT-

229) or MTT assay (for GS-2) at d10 after exposure to either IFN-β or TMZ alone, or

combinations at indicated concentrations, with or without ZVAD-fmk (15 µM).

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

Fig. 1

A

IFNAR-1

IFNAR-2

GAPDHLN

-18

D24

7MG

U87

MG

LN-4

28

U13

8MG

T98

G

U25

1MG

LNT-

229

A17

2

LN-3

19

LN-3

08

U37

3MG

GS-7

GS-2

GS-5

GS-9

GS-8

LTL GIC

- 239 bp

- 146 bp

BIFNAR-1 IFNAR-2 IFNAR-1 IFNAR-2 Isotype

K5

62

LN

T-2

29

LN

-18

LN

-30

8G

S-2

GS

-9

MC

F-7

K5

62

LN

T-2

29

LN

-18

GS

-2G

S-5

C

pSTAT-3

STAT-3

GAPDH

0 50 150 300 500

LNT-229

pSTAT-3

STAT-3

GAPDH

5 15 30 60 240

GS-7

IFN

[IU/ml]

min

LNT-229

5 15 30 60 240 min

5 15 30 60 240 min

GS-9

D

MxA

GAPDH

MxA

GAPDH

0 50 150 300 500 IFN

[IU/ml]

LNT-229

LNT-229

0’ 5’ 15’ 30’ 60’ 4h 24h 48h

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

0%

20%

40%

60%

80%

100%

-IFN +IFN -IFN +IFN -IFN +IFN -IFN +IFN

Fig. 2

A

B

G2/M

S

G1

Sub-G1

CGS-2LNT-229

ddH20

IFN-β

[150 IU/ml]

SAL

[µM]

E

LNT-229 LN-18 GS-2 GS-5

F

LC3 A/B-I

LC3 A/B-II

GAPDH

LNT-229 GS-2

IFN

-

[150IU

/ml]

SA

L [4

M]

ddH

2O

IFN

-

[150IU

/ml]

SA

L [4

M]

ddH

2O

D

0

20

40

60

80

1001

Sp

he

res [n

] ***

******

[n]

1000 5000 10000

Cells

+IF

N-β

-IF

N-β

79.5

31

0

20

40

60

80

100

0

20

40

60

80

100

120

***

***

untreated

untreated

IFN-β

[150 IU/ml]

IFN-β

[150 IU/ml]

IFN-

+ -

G H1.8 1.5

1 1

1 1

31 33

+ IFN-β [150 IU/ml]- IFN-β

F1

Sp

he

res [n

]

10

12

A

bso

rba

nce

[%

]

GS-2

GS-5

GS-7

GS-9

GFAP

β -III-

Tubulin

CNPase

Nestin

+ IFN-β [150 IU/ml]- IFN-β

GS-2

2.8% 3.1%

93.8% 0.3%

6.3%

88.1%

5.3%

0.3%

10.9% 5.3%

77.3% 6.5%

2.4%

94.6% 1.5%

1.5%

19.8%

77.9% 0.9%

1.4%

80.3% 1.4%

13.8% 4.5%

40.5%

56.9%

1.5%

1.1%

66.5% 1.1%

31.2% 1.2%

Annexin V Annexin VAnnexin VAnnexin V

+ IF

N-β

-

IFN

-β

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

Fig. 3

A

B

C

1

0.8

0.6

0

0.2

0.4

1

0.8

0.6

0

0.2

0.4

50

40

30

0

10

20

25

20

15

0

5

10

40

30

0

10

20

60

45

0

15

30

mR

NA

exp

ressio

n

(no

rma

lize

d to

co

ntr

ol)

Effe

ct o

f IF

N [%

]E

ffe

ct o

f IF

N [%

]si_Ctrl si_IFNAR-2

si_IFNAR-1 si_IFNAR-2

LN

T-2

29

LN

-18

LNT-229 LN-18

LNT-229 LN-18

**

*

*** ****** ***

*********

*********

2.2

1.32.4

1.1

knockdown

si_Ctrl R2R1 R1+R2

12.5 µM TMZ 500 µM TMZ

5 Gy 5 Gy

si_Ctrl R2R1 R1+R2

si_Ctrl R2R1 R1+R2 si_Ctrl R2R1 R1+R2

knockdown knockdown

knockdown

LNT-229 LN-18LN-18 LNT-229

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

Fig. 4

A

E

D

C

B

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

20

40

60

80

0

20

40

60

80

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

120

20

40

60

80

100

0

0 17 50 150 0 17 50 150 0 17 50 150 0 17 50 150 0 17 50 150

LNT-229 GS-5GS-2LN-18 GS-9

LNT-229 LN-18 GS-2 GS-5GS-9

200 4000 6.25 12.5 25 50 100 800 0 62 125 250 500 1000

0 2 4 6 8 100 62 125 250 500 1000

GS-2 GS-5 GS-9GS-7

GS-2 GS-5 GS-9GS-7GS-2 GS-5 GS-9GS-7

Clo

no

ge

nic

ity [%

]C

lon

og

en

icity [%

]C

lon

og

en

icity [%

]C

lon

og

en

icity [%

]C

lon

og

en

icity [%

]C

lon

og

en

icity [%

]

IFN-β

[IU/ml]

0

1505017

0

- 24h- 4h- 1h

1 Gy

5 Gy3 Gy

IFN-β

[IU/ml]

0

1505017

*****

**

***

****** **

**

**

****

***

***

***

**

*

* **

*

**

*

**

**

**

***

***

***

******

***

***

***

***

******

***

*****

**

**

*

+

+

+

+

+

1 GyCtrl

GS-7 GS-9

GS-5GS-2

TMZ

[µM]

TMZ

[µM]

TMZ

[µM]

TMZ

[µM]

IFN-β [IU/ml]

6.25 µM TMZ6.25 µM TMZ 500 µM TMZ 500 µM TMZ

LNT-229 LN-18 GS-2 GS-5GS-9

***

******

***

***

**

**

**

****

**

*

**

*

*

*

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

Fig. 5

5000

0

1000

2000

3000

4000

800

0

200

400

600

40

0

10

20

30

20

0

4

8

12

16

30

0

10

20

600

0

200

300

400

500

100

600

0

200

400

600

0

200

300

400

500

100

XA

F-1

mR

NA

exp

ressio

n

(no

rma

lize

d to

co

ntr

ol)

TR

AIL

mR

NA

exp

ressio

n

(no

rma

lize

d to

co

ntr

ol)

LNT-229 GS-2

6 h post IFN-β 24 h post IFN-β 6 h post IFN-β 24 h post IFN-β

0 500300150500 50030015050 0 50030015050 0 50030015050

0 500300150500 500300150500 500300150500 50030015050

XAF-1

GAPDH

TRAIL

0 50030015050 0 50030015050 0 500300150500 50030015050 IFN-β

[IU/ml]

IFN-β

[IU/ml]

IFN-β

[IU/ml]

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/

Published OnlineFirst February 13, 2014.Mol Cancer Ther Caroline Happold, Patrick Roth, Manuela Silginer, et al. therapy resistance of glioblastoma stem cells

induces loss of spherogenicity and overcomesβInterferon-

Updated version

10.1158/1535-7163.MCT-13-0772doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2014/02/19/1535-7163.MCT-13-0772.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

.pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/early/2014/02/13/1535-7163.MCT-13-0772To request permission to re-use all or part of this article, use this link

on April 3, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0772

http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-0772http://mct.aacrjournals.org/content/suppl/2014/02/19/1535-7163.MCT-13-0772.DC1http://mct.aacrjournals.org/cgi/alertsmailto:pubs@aacr.orghttp://mct.aacrjournals.org/content/early/2014/02/13/1535-7163.MCT-13-0772http://mct.aacrjournals.org/

Article FileFig.1Fig.2Fig.3Fig.4Fig.5Fig.6